This application relates generally to methods and compositions for improving the sensitivity and specificity of polynucleotide synthesis and more particularly to methods and compositions using non-nucleic acid polyanions for reversibly inhibiting thermostable polymerase, in a temperature dependent manner, during polynucleotide synthesis.
Polynucleotide synthesis techniques, and polymerase chain reaction (PCR) in particular, include some of the most important biotechnological innovations in the fields of molecular and cell biology and biomedical research. Polynucleotide synthesis involves the synthesis of a complementary polynucleotide strand from a template polynucleotide strand, so for example, the information in the template polynucleotide strand directly guides the formation of a complementary polynucleotide strand from its own sequence. In its more complicated state, polynucleotide synthesis, for example PCR, can be used to amplify specific segments of RNA or DNA in a rapid and highly reproducible manner. Saiki, et al. (1988) Science 239:487-491. Applications for PCR have continued to expand from its inception, for example, PCR is now being used to clone from genomic DNA or cDNA, to perform in vitro mutagenesis and engineering of DNA, to genetically fingerprint forensic samples, to detect pathogenic agents like hepatitis C in blood samples, and to perform direct nucleotide sequencing on genomic DNA.
PCR is a rapid procedure for the in vitro enzymatic amplification of target polynucleotides in an exponential manner. Three nucleic acid segments are required to practice a PCR reaction: a double-stranded polynucleotide containing the target nucleic acid sequence for amplification, and a pair of single-stranded oligonucleotide primers that flank that target sequence. An enzyme (thermostable polymerasexe2x80x94functional at elevated temperatures) and the appropriate deoxyribonucleoside triphosphates (dNTPs), as well as a buffer make up the reaction mixture. In use, the primers are mixed with a buffered solution containing the template polynucleotide, the thermostable polymerase, and the dNTPs for all four deoxynucleotides. The solution is heated to a temperature sufficient to denature the double-stranded template polynucleotide, and abruptly cooled to a temperature sufficient to allow the primers to anneal to the sequences flanking the target sequence on the template polynucleotide. The thermostable polymerase recognizes and binds to the primer-template complexes and the temperature is cycled upward to a temperature at which the thermostable polymerase has optimum activity for polynucleotide synthesis. The thermostable polymerase forms a complementary strand to the template polynucleotide and the process of temperature cycling is repeated. Numerous cycles, providing up to millions/billions of the target sequence, can be performed without altering the reaction mixture.
Polynucleotide synthesis at the elevated temperatures used in PCR tends to prevent the non-specific annealing of primers to non-target polynucleotides and thus improves the specificity and sensitivity of the PCR reaction. In order to operate at these elevated temperatures, thermostable polymerases have been isolated from a number of thermophilic bacterium that live at elevated temperatures, for example, in hot springs, next to underwater volcanic vents, etc. Because these enzymes normally operate at high temperatures, their use eliminates the necessity of repetitively adding temperature sensitive polymerases to the PCR reaction after each temperature cycle.
However, although the performance of PCR at elevated temperatures has reduced the level of non-specific annealing of primers to polynucleotide sequences in the reaction mixture, especially at the elevated temperatures required for optimum thermostable polymerase activity, non-specific primer interactions with polynucleotide sequences, and some level of corresponding primer elongation by the thermostable polymerase, does occurs at lower temperatures. The non-specific interactions and activity of the thermophilic polymerase tends to occur even at temperatures as low as 25xc2x0 C., i.e., during the set-up of the PCR reaction mixture at room temperature, especially when a large number of reactions are handled simultaneously. The activity of Taq DNA polymerase, the most frequently used thermostable DNA polymerase, at 30xc2x0 C. is still 12-15% of its full activity at 70xc2x0 C. This problem is especially prevalent in PCR applications having a small number of target polynucleotide sequences in a milieu containing an excess of non-target, i.e., non-specific, DNA and/or RNA. Several approaches have been advanced within the art to minimize these inherent shortcomings in PCR, the most prevalent of which is termed xe2x80x9chot start PCR.xe2x80x9d
The overall approach to hot start PCR reactions is to physically, chemically or biochemically block the polymerization reaction until the reaction reaches a temperature above the optimal annealing temperature of the primers. In this manner the thermostable polymerase is unable to elongate primer-template polynucleotides at temperatures where non-specific primer-template DNA interactions can exist. With regard to physical hot start PCR, the thermostable polymerase, or one of the other critical reaction components, e.g., dNTPS or magnesium ions, is withheld from the reaction until the reaction reaches temperatures in the range of 85xc2x0 C. to 95xc2x0 C. This temperature is sufficiently high enough to not permit even partial hybridization of the primers to the template polynucleotide, i.e., substantially no non-specific primer annealing to polynucleotides. A number of physical blocks can be used to partition the reaction in a heat dependent manner, including, a wax barrier or wax beads with embedded reaction components, which melts at around 55xc2x0 C. to 65xc2x0 C. However, a shortcoming to using these wax barriers/beads is that the melted material remains in the reaction for the duration of the PCR, forming a potential inhibitor for some PCR applications as well as being incompatible with some potential downstream applications of the amplified product. In some cases the barrier can be physically removed from the reaction to accommodate later uses, but the removal increases the risk of sample-to-sample contamination and requires time and energy to accomplish. A second physical hot start PCR technique utilizes a compartmentalized tube in a temperature regulated centrifuge. The components of the PCR reaction are compartmentalized within the tube from a critical component of the PCR reaction, where the components are all brought together by rupturing the compartments of the tube at a certain g force that corresponds to the specific annealing temperatures of the primer-template polynucleotide. This is accomplished by a dedicated centrifuge that regulates g force with rotor temperature. However, this technique requires expensive equipmentxe2x80x94compartmentalized tubes for each PCR reaction and a specialized centrifugexe2x80x94each factor limiting the number of reactions that can be run at one time and effecting the cost of each reaction.
Another way to implement hot start PCR is to use a thermostable polymerase that has been reversibly inactivated by a chemical modification, such as AMPLITAQ GOLD(trademark) DNA polymerase (Birch et al., 1998, U.S. Pat. No. 5,773,258; Ivanov et al., 2001, U.S. Pat. No. 6,183,998). These techniques are generally referred to as chemical hot start PCR. In the most common type of chemical hot start PCR, the thermostable polymerase, mainly Taq DNA polymerase, has been chemically cross-linked to inactivate the enzyme. The cross-linked thermostable polymerase is reactivated by heating the polymerase prior to the reaction for a predetermined amount of time at 95xc2x0 C. and at a specific pH. Moretti et al. (1998) Biotechniques 25:716-725. The optimal pH for the destruction of the cross-links at 95xc2x0 C. is adjusted by using reaction buffers, which have a pH of 8.0 at 25xc2x0 C. However, this buffer pH is suboptimal for the activity of the thermostable polymerase at 65-70xc2x0 C. in the elongation step during PCR. Another major drawback of the technique is that only a fraction of the enzyme is ever re-activated through heating, leaving a substantial part (up to 50%) of the enzyme in a permanently inactive state. Also the degree of chemical modification is difficult to normalize between various polymerase preparations, and therefore provides a source for batch-to-batch variations of the polymerase activity. This has proven to be costly and has proven to be ineffective at polymerizing longer stretches of target nucleic acid sequence. In addition, the investigator is limited to the use of the chemically modified polymerase and therefore the reaction conditions required for that chemically modified polymerase.
A third way of implementing hot start PCR is by combining a monoclonal antibody specific to the thermostable polymerase with the thermostable polymerase before addition to the PCR reaction. This type of hot start PCR can be referred to as hot start PCR by affinity ligand blocking. The antibody binds to the thermostable polymerase at lower temperatures and blocks activity, but is denatured at higher temperatures, thus rendering the polymerase active. Scalice et al. (1994) J. Immun. Methods 172:147-163; Scalice et al. U.S. Pat. No. 5,338,671; Sharkey et al. (1994) Bio/Technology 12:506-509; Kellogg et al. (1994) Biotechniques 16: 1134-1137. A shortcoming of this technique is the large amount of the antibodies (up to 0.6 xcexcg) which must be added to achieve efficient inhibition of the thermostable DNA polymerase. Once the antibodies are denatured, they tend to cause some level of inhibition on the thermophilic polymerase activity even at higher temperatures. The high concentration of denatured protein reduces the product yield of PCR. Recently, the immunoglobulin fraction has been identified as the major PCR inhibitor in DNA preparations from blood cells. Al-Soud W. A. and Radstroem P., J Clin. Microbiol. 2001, 39(2):485-493. Also, as above, the investigator is limited to the monoclonal antibody targeted thermostable polymerase. Each type of thermostable DNA polymerase requires its own technical solution.
An alternative ligand blocking hot start PCR technique has been developed based on aptamers, single-stranded oligonucleotides possessing a DNA sequence with high binding affinity for the active center of selected thermophilic DNA polymerases (Gold et al. 2000, U.S. Pat. No. 6,020,130; Jayasena et al. 2001, U.S. Pat. No. 6,183,967). The single-stranded oligonucleotides bind to the thermophilic polymerase at lower temperatures but are released at higher temperatures. However, as above for the monoclonal antibody technique, a large excess of oligonucleotide, up to 0.1 xcexcg/rxn, is necessary to inhibit the polymerasexe2x80x94adding a significant cost to each reaction. Further, the single-stranded oligonucleotides can themselves act as primers and non-specific targets for the thermophilic polymerase, adding to the level of potential non-specific products. Expensive chemical modifications of the 3xe2x80x2-ends of the oligonucleotide aptamers are necessary to prevent this. Note also, that this technique has been extended to using short double stranded oligonucleotides with a specific xe2x80x9cstem-loopxe2x80x9d secondary structure rather than a specific DNA sequence to block the active center of thermostable DNA polymerases. However, as above, the additional costs per reaction are significant, and the potential of non-specific priming has to be eliminated by modification of the 3xe2x80x2-terminal hydroxyl group. Although, these technologies have never been utilized for a commercial product, they demonstrated the usefulness of competitive inhibitors which resemble the structure of the template polynucleotide to prevent unwanted binding of the DNA substrate to the thermostable DNA polymerase. In contrast to all previously discussed techniques for hot start PCR, competitive oligonucleotide aptamers provide permanent control over non-specific primer binding and extension throughout the PCR and not only prior to the first PCR cycle.
Finally and more recently, another hot start PCR approach has been developed that depends on using a genetically engineered Taq DNA polymerase having mutations that renders the enzyme inactive below 35xc2x0 C. (Barnes et al.; 2001; U.S. Pat. No. 6,214,557). However, this N-terminally truncated form of Taq DNA polymerase has about a five times lower processivity than wild type enzyme, thereby requiring a 5-10 fold activity excess of the enzyme in each PCR reaction as compared to the wild type Taq DNA polymerase. As a result, this technique is limited to the amplification of short target sequences less than 1 kb. In addition this technique did not provide data as to whether the mutations causing inactivation of the truncated Taq polymerase at low temperatures could submit the same effect to the full-size enzyme.
The present invention uses for the first time strong polyanionic polymerase inhibitors to control the activity of thermostable DNA polymerases in dependence on the applied incubation temperature. The inhibitory effect of natural and synthetic polyanions (Holler et al., 1992, Holler et al., Shimada et al., 1978), in particular of sulfated polysaccharides (Hitzeman et al., 1978), on various DNA and RNA polymerases (Ferencz et al., 1975) is well known for many years. It has also been found that homopolymeric stretches of DNA as a special case of a natural polyanion possess inhibitory activity on several types of eucaryotic DNA polymerases (Shimada et al., 1978). Recently, Kainz et al. (Kainz et al., (2000) Biochim Biophys Acta 28(2):278-82) described the inhibition of PCR by the addition non-specific double stranded DNA from E. coli phage lambda. Kainz and coworkers proposed a mechanism for DNA inhibition where an excess of non-specific ds DNA binds out the available active Taq DNA polymerase and argued that this feature of Taq polymerase provides the reason for saturation of the PCR amplification reaction during late cycles. The available free enzyme is bound out by the accumulated ds PCR product. This effect was utilized to inhibit Taq DNA polymerase at ambient temperatures with an excess of small ds oligonucleotides (Kainz et al., (2000) Biotechniques 15:1494(1-2):23-7).
Acid polyanionic polysaccharides have been characterized as the major PCR inhibitor in plant DNA isolations (Demeke et al., 1992), whereas sulfated polysaccharadies, such as dextran sulfate and heparin were identified as potent PCR inhibitors contaminating DNA preparations from blood cells (Al-Soud et al., 2001). Sulfated polysaccharides in particular show a broad spectrum of inhibition against a variety of DNA-modifying enzymes including Polynucleotide Kinase (Wu et al., 1971), restriction endonucleases (Do et al., 1991) and retroviral reverse transcriptases (Moelling et al., 1989). Although the inhibitory effect of polyanions and sulfated polysaccharides in particular has been studied for many years, the exact mechanism is not known (Furukawa et al., 1983). Also the factors determining the degree of inhibition other than the concentration of the polyanion have not been studied systematically yet. It is generally suggested that anionic polysaccharides are competitive inhibitors of DNA- and RNA modifying enzymes competing with the substrate nucleic acids for binding the enzyme. The chemical structure of anionic acidic polysaccharides resembles the polyp entose phosphate structure of the backbone of nucleic acids. Based on this principle DNA and RNA polymerases, DNA binding enzymes, and in particular, Taq DNA polymerase are purified by affinity chromatography on heparin Sepharose. Recently, several single amino acid substitutions both on the polymerase and N-terminal exonuclease domain of Taq DNA polymerase have been found to drastically reduce the susceptibility of Taq DNA polymerase for inhibition by heparin (Ghadessy et al., 2001). This represents the first direct experimental indication that the inhibitive effect of heparin is related to binding of this sulfated polysaccharide to certain sites of the DNA polymerase molecule.
The binding affinity of any ligand to its polymerase depends not only on its molar concentration in relation to the amount of the polymerase, but on many other parameters too, such as temperature, molecular weight (size), density of charged groups on the surface of the molecule, the concentration of a competing nucleic acid substrates and the concentration of counter ions (if ionic interactions play the major role in ligand-polymerase binding). In the past only the inhibitive concentrations of polyanions has been investigated. The present invention did for the first time systematically study the influence of the other parameters on the inhibition of DNA polymerase by sulfated polysaccharides. The use of a thermostable polymerase did provide the opportunity to study the effect of increasing temperature on the polyanion inhibition. Surprisingly, conditions and parameters were found under which the strong inhibition of polynucleotide synthesis by sulfated polysaccharides is getting reversible with increasing temperatures. The following parameters have been identified as critical for the use of sulfated polysaccharides for thermoreversible inhibition of a thermostable DNA polymerase: using low molecular weight (small sized) versus high molecular weight polyanions, working in a nanomolar concentration range in the polymerization reaction mixture, balancing the polyanions with an appropriate concentration of monovalent counter ions.
Also the detailed experiments in the present invention have been performed with dextran sulfate derivatives and heparin and Taq DNA polymerase, a person with skills in the art can use the results and techniques disclosed in the present invention to adapt the disclosed method for inhibitive complexes of other sulfated polysaccharides or polyanions of similar characteristics with other thermostable polymerases for thermoreversible inhibition of the polymerase activity.
Against this background the present invention has been developed.
The present invention uses for the first time strong polyanionic polymerase inhibitors to control the activity of thermostable DNA polymerases dependent on the applied incubation temperature.
The present invention includes methods and compositions for the temperature dependent inhibition of thermostable polymerases during polynucleotide synthesis. Temperature stability (at least up to 60xc2x0 C.) of the polymerases used is important, because the temperature is the major parameter to control the binding affinity of the competitive polymerase inhibitors used.
In one aspect, the present invention is a method of polynucleotide synthesis that includes combining in a polymerization buffer having 35-60 mM monovalent cations and at least 1.5 mM magnesium ions, a thermostable polymerase, at least one dNTP, and a non-nucleic acid polyanion prior to the addition of a template nucleic acid molecule hybridized with appropriate primers to the template nucleic acid molecule. The temperature of the reaction mixture and the concentration of the non-nucleic acid polyanion are set at values at which the non-nucleic acid polyanion competitively inhibits the binding of the thermostable polymerase to the primed template polynucleotide substrate. The reaction mixture is then heated to a temperature at which the non-nucleic acid polyanion dissociates from the thermostable polymerase, thereby permitting the binding and subsequent elongation of the primed template polynucleotide substrate.
In another aspect, the present invention is a composition for polynucleotide synthesis, including a thermostable polymerase, a non-nucleic acid polyanion, a template nucleic acid molecule, primers, dNTPs and a reaction buffer having 35-60 mM monovalent cations.